Pesticidal toxins

ABSTRACT

The subject invention concerns new classes of insecticidal proteins obtainable from  Bacillus thuringiensis , and polynucleotides that encode these proteins. The subject invention also includes transgenic cells and plants that produce these proteins. The proteins are preferably in the 10-15 kDa and 40-50 kDa size range.

This application is a continuation of U.S. application Ser. No.09/548,334, filed Apr. 12, 2000, now U.S. Pat. No. 6,548,291, and also acontinuation of U.S. application Ser. No. 09/547,621, filed Apr. 12,2000, now U.S. Pat. No. 6,624,145; Ser. Nos. 09/548,334 and 09/574,621are divisionals of U.S. application Ser. No. 08/844,188, filed Apr. 18,1997, now U.S. Pat. No. 6,127,180; which is a continuation-in-part ofU.S. application Ser. No. 08/633,993, filed Apr. 19, 1996, now U.S. Pat.No. 6,083,499.

BACKGROUND OF THE INVENTION

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive,spore-forming bacterium characterized by parasporal crystalline proteininclusions. These inclusions often appear microscopically asdistinctively shaped crystals. The proteins can be highly toxic to pestsand specific in their toxic activity. Certain B.t. toxin genes have beenisolated and sequenced, and recombinant DNA-based B.t. products havebeen produced and approved for use. In addition, with the use of geneticengineering techniques, new approaches for delivering these B.t.endotoxins to agricultural environments are under development, includingthe use of plants genetically engineered with endotoxin genes for insectresistance and the use of stabilized intact microbial cells as B.t.endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commerciallyvaluable.

Until the last ten years, commercial use of B.t. pesticides has beenlargely restricted to a narrow range of lepidopteran (caterpillar)pests. Preparations of the spores and crystals of B. thuringiensissubsp. kurstaki have been used for many years as commercial insecticidesfor lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1produces a crystalline δ-endotoxin which is toxic to the larvae of anumber of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticideswith specificities for a much broader range of pests. For example, otherspecies of B.t., namely israelensis and tenebrionis (a.k.a. B.t. M-7,a.k.a. B.t. san diego), have been used commercially to control insectsof the orders Diptera and Coleoptera, respectively (Gaertner, F. H.[1989] “Cellular Delivery Systems for Insecticidal Proteins: Living andNon-Living Microorganisms,” in Controlled Delivery of Crop ProtectionAgents, R. M. Wilkins, ed., Taylor and Francis, New York and London,1990, pp. 245-255). See also Couch, T. L. (1980) “Mosquito Pathogenicityof Bacillus thuringiensis var. israelensis,” Developments in IndustrialMicrobiology 22:61-76; Beegle, C. C., (1978) “Use of EntomogenousBacteria in Agroecosystems,” Developments in Industrial Microbiology20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter(1983) Z. ang. Ent. 96:500-508, describe Bacillus thuringiensis var.tenebrionis, which is reportedly active against two beetles in the orderColeoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni.

Recently, new subspecies of B.t. have been identified, and genesresponsible for active δ-endotoxin proteins have been isolated (Höfte,H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Höfteand Whiteley classified B.t. crystal protein genes into 4 major classes.The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- andDiptera-specific), CryIII (Coleoptera-specific), and CryIV(Diptera-specific). The discovery of strains specifically toxic to otherpests has been reported. (Feitelson, J. S., J. Payne, L. Kim [1992]Bio/Technology 10:271-275).

The cloning and expression of a B.t. crystal protein gene in Escherichiacoli has been described in the published literature (Schnepf, H. E., H.R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat.No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expressionof B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,797,276 and4,853,331 disclose B. thuringiensis strain tenebrionis (a.k.a. M-7,a.k.a. B.t. san diego) which can be used to control coleopteran pests invarious environments. U.S. Pat. No. 4,918,006 discloses B.t. toxinshaving activity against Dipterans. U.S. Pat. No. 4,849,217 disclosesB.t. isolates which have activity against the alfalfa weevil. U.S. Pat.No. 5,208,077 discloses coleopteran-active Bacillus thuringiensisisolates. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclosecertain isolates of B.t. which have activity against nematodes. As aresult of extensive research and investment of resources, other patentshave issued for new B.t. isolates and new uses of B.t. isolates.However, the discovery of new B.t. isolates and new uses of known B.t.isolates remains an empirical, unpredictable art.

Coleopterans are an important group of agricultural pests which cause avery large amount of damage each year. Examples of coleopteran pestsinclude alfalfa weevils and corn rootworm.

The alfalfa weevil, Hypera postica, and the closely related Egyptianalfalfa weevil, Hypera brunneipennis, are the most important insectpests of alfalfa grown in the United States, with 2.9 million acresinfested in 1984. An annual sum of 20 million dollars is spent tocontrol these pests. The Egyptian alfalfa weevil is the predominantspecies in the southwestern U.S., where it undergoes aestivation (i.e.,hibernation) during the hot summer months. In all other respects, it isidentical to the alfalfa weevil, which predominates throughout the restof the U.S.

The larval stage is the most damaging in the weevil life cycle. Byfeeding at the alfalfa plant's growing tips, the larvae causeskeletonization of leaves, stunting, reduced plant growth, and,ultimately, reductions in yield. Severe infestations can ruin an entirecutting of hay. The adults, also foliar feeders, cause additional, butless significant, damage.

Approximately 9.3 million acres of U.S. corn are infested with cornrootworm species complex each year. The corn rootworm species complexincludes the northern corn rootworm, Diabrotica barberi, the southerncorn rootworm, D. undecimpunctata howardi, and the western cornrootworm, D. virgifera virgifera. The soil-dwelling larvae of theseDiabrotica species feed on the root of the corn plant, causing lodging.Lodging eventually reduces corn yield and often results in death of theplant. By feeding on cornsilks, the adult beetles reduce pollinationand, therefore, detrimentally effect the yield of corn per plant. Inaddition, adults and larvae of the genus Diabrotica attack cucurbitcrops (cucumbers, melons, squash, etc.) and many vegetable and fieldcrops in commercial production as well as those being grown in homegardens.

Control of corn rootworm has been partially addressed by cultivationmethods, such as crop rotation and the application of high nitrogenlevels to stimulate the growth of an adventitious root system. However,chemical insecticides are relied upon most heavily to guarantee thedesired level of control. Insecticides are either banded onto orincorporated into the soil. The major problem associated with the use ofchemical insecticides is the development of resistance among the treatedinsect populations.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns novel materials and methods forcontrolling non-mammalian pests. In a preferred embodiment, the subjectinvention provides materials and methods for the control of coleopteranpests. In specific embodiments, the materials and methods describedherein are used to control alfalfa weevil and/or corn rootworm.

The subject invention advantageously provides two new classes ofpolynucleotide sequences which encode corresponding novel classes ofpesticidal proteins. One novel class of polynucleotide sequences asdescribed herein encodes toxins which have a full-length molecularweight of approximately 40-50 kDa. In a specific embodiment, thesetoxins have a molecular weight of about 43-47 kDa. A second class ofpolynucleotides, which encodes pesticidal proteins of about 10-15 kDa,is also provided according to the subject invention. In a specificembodiment, these toxins have a molecular weight of about 13-14 kDa. Thesubject invention concerns polynucleotides which encode the 40-50 kDaand 10-15 kDa toxins, polynucleotides which encode pesticidal fragmentsof the full length toxins, and polynucleotide sequences which encodelonger forms of these toxins which include, for example, a protoxinregion. In a preferred embodiment, these toxins, including thefragments, are active against coleopteran pests.

Specific B.t. toxins useful according to the invention include toxinswhich can be obtained from the B.t. isolates designated as PS80JJ1,PS149B1, and PS167H2. Of these, PS149B1 and PS167H2 are novel isolates.The subject invention also includes the use of variants of theexemplified B.t. isolates and toxins which have substantially the samecoleopteran-active properties as the specifically exemplified B.t.isolates and toxins. Such variant isolates would include, for example,mutants. Procedures for making mutants are well known in themicrobiological art. Ultraviolet light and chemical mutagens such asnitrosoguanidine are used extensively toward this end.

In one embodiment of the subject invention, the polynucleotide sequencesof the subject invention are used to encode toxins of approximately43-47 kDa. These toxins are then used to control coleopteran pests. In aparticularly preferred embodiment, the coleopteran pests are cornrootworms. The genes which encode the 43-47 kDa toxins can be obtainedfrom, for example, PS80JJ1, PS149B1, or PS167H2. In a second embodiment,toxins of approximately 13-14 kDa are used to control coleopteran pests.The approximately 13-14 kDa toxin, as well as the genes which encodethese toxins, can also be obtained from PS80JJ1, PS149B1, or PS167H2. Ina particularly preferred embodiment, the activity of the 43-47 kDatoxins can be augmented and/or facilitated by further contacting thetarget pests with an approximately 13-14 kDa toxin.

In a preferred embodiment, the subject invention concerns plants cellstransformed with at least one polynucleotide sequence of the subjectinvention such that the transformed plant cells express pesticidaltoxins in tissues consumed by the target pests.

Alternatively, the B.t. isolates of the subject invention, orrecombinant microbes expressing the toxins described herein, can be usedto control pests. In this regard, the invention includes the treatmentof substantially intact B.t. cells, and/or recombinant cells containingthe expressed toxins of the invention, treated to prolong the pesticidalactivity when the substantially intact cells are applied to theenvironment of a target pest. The treated cell acts as a protectivecoating for the pesticidal toxin. The toxin becomes active uponingestion by a target insect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows three specific 43-47 kDa pesticidal toxins of the subjectinvention as well as a consensus sequence for these pesticidal toxins.

FIG. 2 shows the relationship of the 14 and 45 kDa sequences of PS80JJ1(SEQ ID NOS. 31 and 10).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO.1 is a 5-amino acid N-terminal sequence of the approximately45 kDa toxin of 80JJ1.

SEQ ID NO.2 is a 25-amino acid N-terminal sequence of the approximately45 kDa toxin of 80JJ1.

SEQ ID NO.3 is a 24-amino acid N-terminal sequence of the approximately14 kDa toxin of 80JJ1.

SEQ ID NO.4 is the N-terminal sequence of the approximately 47 kDa toxinfrom 149B1.

SEQ ID NO.5 is a 50-amino acid N-terminal amino acid sequence for thepurified approximately 14 kDa protein from PS149B1.

SEQ ID NO.6 is the N-terminal sequence of the approximately 47 kDa toxinfrom 167H2.

SEQ ID NO. 7 is a 25-amino acid N-terminal sequence for the purifiedapproximately 14 kDa protein from PS167H2.

SEQ ID NO.8 is an oligonucleotide probe for the gene encoding thePS80JJ1 44.3 kDa toxin and is a forward primer for PS149B1 and PS167H2used according to the subject invention.

SEQ ID NO.9 is a reverse primer for PS149B1 and PS167H2 used accordingto the subject invention.

SEQ ID NO.10 is the nucleotide sequence of the gene encoding theapproximately 45 kDa PS80JJ1 toxin.

SEQ ID NO.11 is the amino acid sequence for the approximately 45 kDaPS80JJ1 toxin.

SEQ ID NO. 12 is the partial nucleotide sequence of the gene encodingthe approximately 44 kDa PS149B1 toxin.

SEQ ID NO.13 is the partial amino acid sequence for the approximately 44kDa PS149B1 toxin.

SEQ ID NO. 14 is the partial nucleotide sequence of the gene encodingthe approximately 44 kDa PS167H2 toxin.

SEQ ID NO. 15 is the partial amino acid sequence for the approximately44 kDa PS167H2 toxin.

SEQ ID NO.16 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO.17 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO.18 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO.19 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO.20 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO. 16.

SEQ ID NO.21 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO. 17.

SEQ ID NO.22 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO. 18.

SEQ ID NO.23 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO. 19.

SEQ ID NO.24 is a reverse primer based on the reverse complement of SEQID NO. 22.

SEQ ID NO.25 is a reverse primer based on the reverse complement of SEQID NO. 23.

SEQ ID NO.26 is a forward primer based on the PS80JJ1 44.3 kDa toxin.

SEQ ID NO.27 is a reverse primer based on the PS80JJ1 44.3 kDa toxin.

SEQ ID NO.28 is a generic sequence representing a new class of toxinsaccording to the subject invention.

SEQ ID NO.29 is an oligonucleotide probe used according to the subjectinvention.

SEQ ID NO. 30 is the nucleotide sequence of the entire genetic locuscontaining open reading frames of both the 14 and 45 kDa PS80JJ1 toxinsand the flanking nucleotide sequences.

SEQ ID NO. 31 is the nucleotide sequence of the PS80JJ1 14 kDa toxinopen reading frame.

SEQ ID NO.32 is the deduced amino acid sequence of the 14 kDa toxin ofPS80JJ1.

SEQ ID NO. 33 is a reverse oligonucleotide primer used according to thesubject invention.

SEQ ID NO. 34 is the nucleotide sequence of the entire genetic locuscontaining open reading frames of both the 14 and 44 kDa PS167H2 toxinsand the flanking nucleotide sequences.

SEQ ID NO. 35 is the nucleotide sequence of the gene encoding theapproximately 14 kDa PS167H2 toxin.

SEQ ID NO. 36 is the amino acid sequence for the approximately 14 kDaPS167H2 toxin.

SEQ ID NO. 37 is the nucleotide sequence of the gene encoding theapproximately 44 kDa PS167H2 toxin.

SEQ ID NO. 38 is the amino acid sequence for the approximately 44 kDaPS167H2 toxin.

SEQ ID NO. 39 is the nucleotide sequence of the entire genetic locuscontaining open reading frames of both the 14 and 44 kDa PS149B1 toxinsand the flanking nucleotide sequences.

SEQ ID NO. 40 is the nucleotide sequence of the gene encoding theapproximately 14 kDa PS149B1 toxin.

SEQ ID NO. 41 is the amino acid sequence for the approximately 14 kDaPS149B1 toxin.

SEQ ID NO. 42 is the nucleotide sequence of the gene encoding theapproximately 44 kDa PS149B1 toxin.

SEQ ID NO. 43 is the amino acid sequence for the approximately 44 kDaPS149B1 toxin.

SEQ ID NO. 44 is a maize-optimized gene sequence encoding theapproximately 14 kDa toxin of 80JJ1.

SEQ ID NO. 45 is a maize-optimized gene sequence encoding theapproximately 44 kDa toxin of 80JJ1.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns two new classes of polynucleotidesequences which encode novel pesticidal toxins. In one embodiment, thetoxins have a full-length molecular weight of approximately 40-50 kDa.In specific embodiments exemplified herein, these toxins have amolecular weight of about 43-47 kDa. In a second embodiment, thepesticidal toxins have a molecular weight of approximately 10-15 kDa. Inspecific embodiments exemplified herein, these toxins have a molecularweight of about 13-14 kDa. Certain specific toxins are exemplifiedherein. For toxins having a known amino acid sequence, the molecularweight is also known. Those skilled in the art will recognize that theapparent molecular weight of a protein as determined by gelelectrophoresis will sometimes differ from the true molecular weight.Therefore, reference herein to, for example, a 45 kDa protein or a 14kDa protein is understood to refer to proteins of approximately thatsize even if the true molecular weight is somewhat different.

The subject invention concerns not only the polynucleotide sequenceswhich encode these classes of toxins, but also the use of thesepolynucleotide sequences to produce recombinant hosts which express thetoxins. In a further aspect, the subject invention concerns the combineduse of an approximately 40-50 kDa toxin of the subject inventiontogether with an approximately 10-15 kDa toxin of the subject inventionto achieve highly effective control of pests, including coleopteranssuch as corn rootworm.

A further aspect of the subject invention concerns two novel isolatesand the toxins and genes obtainable from these isolates. The novel B.t.isolates of the subject invention have been designated PS149B1 andPS167H2.

The new classes of toxins and polynucleotide sequences provided here aredefined according to several parameters. One critical characteristic ofthe toxins described herein is pesticidal activity. In a specificembodiment, these toxins have activity against coleopteran pests. Thetoxins and genes of the subject invention can be further defined bytheir amino acid and nucleotide sequences. The sequences of themolecules within each novel class can be defined herein in terms ofhomology to certain exemplified sequences as well as in terms of theability to hybridize with, or be amplified by, certain exemplifiedprobes and primers. The classes of toxins provided herein can also beidentified based on their immunoreactivity with certain antibodies andbased upon their adherence to a generic formula.

The sequence of three approximately 45 kDa toxins of the subjectinvention are provided as SEQ ID NOS. 11, 43, and 38. In a preferredembodiment of the subject invention, the toxins in this new class have asequence which conforms to the generic sequence presented as SEQ ID NO.28. In a specific embodiment, the toxins of this class will conform tothe consensus sequence shown in FIG. 1.

In a preferred embodiment, the toxins of the subject invention have atleast one of the following characteristics:

-   -   (a) said toxin is encoded by a nucleotide sequence which        hybridizes under stringent conditions with a nucleotide sequence        selected from the group consisting of: DNA which encodes SEQ ID        NO. 2, DNA which encodes SEQ ID NO. 4, DNA which encodes SEQ ID        NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, DNA which encodes SEQ ID NO.        11, SEQ ID NO. 12, DNA which encodes SEQ ID NO. 13, SEQ ID NO.        14, DNA which encodes SEQ ID NO. 15, DNA which encodes SEQ ID        NO. 16, DNA which encodes SEQ ID NO. 17, DNA which encodes SEQ        ID NO. 18, DNA which encodes SEQ ID NO. 19, SEQ ID NO. 20, SEQ        ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID        NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, DNA which encodes a        pesticidal portion of SEQ ID NO. 28, SEQ ID NO. 37, DNA which        encodes SEQ ID NO. 38, SEQ ID NO. 42, and DNA which encodes SEQ        ID NO. 43;    -   (b) said toxin immunoreacts with an antibody to an approximately        40-50 kDa pesticidal toxin, or a fragment thereof, from a        Bacillus thuringiensis isolate selected from the group        consisting of PS80JJ1 having the identifying characteristics of        NRRL B-18679, PS149B1 having the identifying characteristics of        NRRL B-21553, and PS167H2 having the identifying characteristics        of NRRL B-21554;    -   (c) said toxin is encoded by a nucleotide sequence wherein a        portion of said nucleotide sequence can be amplified by PCR        using a primer pair selected from the group consisting of SEQ ID        NOS. 20 and 24 to produce a fragment of about 495 bp, SEQ ID        NOS. 20 and 25 to produce a fragment of about 594 bp, SEQ ID        NOS. 21 and 24 to produce a fragment of about 471 bp, and SEQ ID        NOS. 21 and 25 to produce a fragment of about 580 bp;    -   (d) said toxin comprises a pesticidal portion of the amino acid        sequence shown in SEQ ID NO. 28;    -   (e) said toxin comprises an amino acid sequence which has at        least about 60% homology with a pesticidal portion of an amino        acid sequence selected from the group consisting of SEQ ID NO.        11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 38, and SEQ ID NO.        43;    -   (f) said toxin is encoded by a nucleotide sequence which        hybridizes under stringent conditions with a nucleotide sequence        selected from the group consisting of DNA which encodes SEQ ID        NO. 3, DNA which encodes SEQ ID NO. 5, DNA which encodes SEQ ID        NO. 7, DNA which encodes SEQ ID NO. 32, DNA which encodes SEQ ID        NO. 36, and DNA which encodes SEQ ID NO. 41;    -   (g) said toxin immunoreacts with an antibody to an approximately        10-15 kDa pesticidal toxin, or a fragment thereof, from a        Bacillus thuringiensis isolate selected from the group        consisting of PS80JJ1 having the identifying characteristics of        NRRL B-18679, PS149B1 having the identifying characteristics of        NRRL B-21553, and PS167H2 having the identifying characteristics        of NRRL B-21554;    -   (h) said toxin is encoded by a nucleotide sequence wherein a        portion of said nucleotide sequence can be amplified by PCR        using the primer pair of SEQ ID NO. 29 and SEQ ID NO. 33; and    -   (i) said toxin comprises an amino acid sequence which has at        least about 60% homology with an amino acid sequence selected        from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID        NO. 7, pesticidal portions of SEQ ID NO. 32, pesticidal portions        of SEQ ID NO. 36, and pesticidal portions of SEQ ID NO. 41.

As used herein “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with 32P-labeled gene-specific probes was performed by standardmethods (Maniatis, T., E. F. Fritsch, J. Sambrook [1982] MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). In general, hybridization and subsequent washes werecarried out under stringent conditions that allowed for detection oftarget sequences with homology to the PS80JJ1 toxin genes. Fordouble-stranded DNA gene probes, hybridization was carried out overnightat 20-25° C. below the melting temperature (Tm) of the DNA hybrid in6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Themelting temperature is described by the following formula (Beltz, G. A.,K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983]Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] AcademicPress, New York 100:266-285).

-   -   Tm=81.5° C.+16.6 Log[Na+]+0.41(%G+C)−0.61(%formamide)−600/length        of duplex in base pairs.

Washes are typically carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at Tm−20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula:Tm (° C.)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura,and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes,D. D. Brown [ed.], Academic Press, New York, 23:683-693).

Washes were typically carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

With the teachings provided herein, one skilled in the art could readilyproduce and use the various toxins and polynucleotide sequences of thenovel classes described herein.

Microorganisms useful according to the subject invention have beendeposited in the permanent collection of the Agricultural ResearchService Patent Culture Collection (NRRL), Northern Regional ResearchCenter, 1815 North University Street, Peoria, Ill. 61604, USA. Theculture repository numbers of the deposited strains are as follows:

Culture Repository No. Deposit Date B.t. strain PS80JJ1 NRRL B-18679Jul. 17, 1990 B.t. strain PS149B1 NRRL B-21553 Mar. 28, 1996 B.t. strainPS167H2 NRRL B-21554 Mar. 28, 1996 E. coli NM522 (pMYC2365) NRRL B-21170Jan. 5, 1994 E. coli NM522 (pMYC2382) NRRL B-21329 Sep. 28, 1994 E. coliNM522 (pMYC2379) NRRL B-21155 Nov. 3, 1993 E. coli NM522 (pMYC2421) NRRLB-21555 Mar. 28, 1996 E. coli NM522 (pMYC2427) NRRL B-21672 Mar. 26,1997 E. coli NM522 (pMYC2429) NRRL B-21673 Mar. 26, 1997 E. coli NM522(pMYC2426) NRRL B-21671 Mar. 26, 1997The PS80JJ1 isolate is available to the public by virtue of the issuanceof U.S. Pat. No. 5,151,363.

B.t. isolates PS149B1 and PS167H2 have been deposited under conditionsthat assure that access to the cultures will be available during thependency of this patent application to one determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37CFR 1.14 and 35 U.S.C. 122. The deposits are available as required byforeign patent laws in countries wherein counterparts of the subjectapplication, or its progeny, are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of a deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposit(s) should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Following is a table which provides characteristics of certain B.t.isolates useful according to the subject invention.

TABLE 1 Description of B.t. strains toxic to coleopterans CrystalApprox. MW NRRL Deposit Culture Description (kDa) Serotype Deposit DatePS80JJ1 multiple 130, 90, 47, 37, 14 4a4b, sotto B-18679 Jul. 17, 1990attached PS149B1 130, 47, 14 B-21553 Mar. 28, 1996 PS167H2  70, 47, 14B-23554 Mar. 28, 1996

Genes and toxins. The genes and toxins useful according to the subjectinvention include not only the full length sequences disclosed but alsofragments of these sequences, variants, mutants, and fusion proteinswhich retain the characteristic pesticidal activity of the toxinsspecifically exemplified herein. As used herein, the terms “variants” or“variations” of genes refer to nucleotide sequences which encode thesame toxins or which encode equivalent toxins having pesticidalactivity. As used herein, the term “equivalent toxins” refers to toxinshaving the same or essentially the same biological activity against thetarget pests as the claimed toxins.

It should be apparent to a person skilled in this art that genesencoding active toxins can be identified and obtained through severalmeans. The specific genes exemplified herein may be obtained from theisolates deposited at a culture depository as described above. Thesegenes, or portions or variants thereof, may also be constructedsynthetically, for example, by use of a gene synthesizer. Variations ofgenes maybe readily constructed using standard techniques for makingpoint mutations. Also, fragments of these genes can be made usingcommercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Also, genes which encode active fragments may beobtained using a variety of restriction enzymes. Proteases may be usedto directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can bederived from B.t. isolates and/or DNA libraries using the teachingsprovided herein. There are a number of methods for obtaining thepesticidal toxins of the instant invention. For example, antibodies tothe pesticidal toxins disclosed and claimed herein can be used toidentify and isolate other toxins from a mixture of proteins.Specifically, antibodies may be raised to the portions of the toxinswhich are most constant and most distinct from other B.t. toxins. Theseantibodies can then be used to specifically identify equivalent toxinswith the characteristic activity by immunoprecipitation, enzyme linkedimmunosorbent assay (ELISA), or western blotting. Antibodies to thetoxins disclosed herein, or to equivalent toxins, or fragments of thesetoxins, can readily be prepared using standard procedures in this art.The genes which encode these toxins can then be obtained from themicroorganism.

Fragments and equivalents which retain the pesticidal activity of theexemplified toxins would be within the scope of the subject invention.Also, because of the redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate these alternative DNA sequences encoding the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention. As used herein, reference to “essentially thesame” sequence refers to sequences which have amino acid substitutions,deletions, additions, or insertions which do not materially affectpesticidal activity. Fragments retaining pesticidal activity are alsoincluded in this definition.

A further method for identifying the toxins and genes of the subjectinvention is through the use of oligonucleotide probes. These probes aredetectable nucleotide sequences. These sequences may be detectable byvirtue of an appropriate label or may be made inherently fluorescent asdescribed in International Application No. WO93/16094. As is well knownin the art, if the probe molecule and nucleic acid sample hybridize byforming a strong bond between the two molecules, it can be reasonablyassumed that the probe and sample have substantial homology. Preferably,hybridization is conducted under stringent conditions by techniqueswell-known in the art, as described, for example, in Keller, G. H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.Detection of the probe provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying toxin-encoding genes of thesubject invention. The nucleotide segments which are used as probesaccording to the invention can be synthesized using a DNA synthesizerand standard procedures. These nucleotide sequences can also be used asPCR primers to amplify genes of the subject invention.

Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid homology with an exemplified toxin. The amino acididentity will typically be greater than 60%, preferably be greater than75%, more preferably greater than 80%, more preferably greater than 90%,and can be greater than 95%. The amino acid homology will be highest incritical regions of the toxin which account for biological activity orare involved in the determination of three-dimensional configurationwhich ultimately is responsible for the biological activity. In thisregard, certain amino acid substitutions are acceptable and can beexpected if these substitutions are in regions which are not critical toactivity or are conservative amino acid substitutions which do notaffect the three-dimensional configuration of the molecule. For example,amino acids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 2 provides a listing of examples of amino acidsbelonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

The toxins of the subject invention can also be characterized in termsof the shape and location of toxin inclusions, which are describedabove.

Recombinant hosts. The toxin-encoding genes harbored by the isolates ofthe subject invention can be introduced into a wide variety of microbialor plant hosts. Expression of the toxin gene results, directly orindirectly, in the intracellular production and maintenance of thepesticide. With suitable microbial hosts, e.g., Pseudomonas, themicrobes can be applied to the situs of the pest, where they willproliferate and be ingested. The result is a control of the pest.Alternatively, the microbe hosting the toxin gene can be treated underconditions that prolong the activity of the toxin and stabilize thecell. The treated cell, which retains the toxic activity, then can beapplied to the environment of the target pest.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing a B.t. geneencoding a toxin into a microorganism host under conditions which allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Control of coleopterans, including corn rootworm using the isolates,toxins, and genes of the subject invention can be accomplished by avariety of methods known to those skilled in the art. These methodsinclude, for example, the application of B.t. isolates to the pests (ortheir location), the application of recombinant microbes to the pests(or their locations), and the transformation of plants with genes whichencode the pesticidal toxins of the subject invention. Recombinantmicrobes may be, for example, a B.t., E. coli, or Pseudomonas.Transformations can be made by those skilled in the art using standardtechniques. Materials necessary for these transformations are disclosedherein or are otherwise readily available to the skilled artisan.

Synthetic genes which are functionally equivalent to the toxins of thesubject invention can also be used to transform hosts. Methods for theproduction of synthetic genes can be found in, for example, U.S. Pat.No. 5,380,831.

Control of other pests such as lepidopterans and other insects,nematodes, and mites can also be accomplished by those skilled in theart using standard techniques combined with the teachings providedherein.

Treatment of cells. As mentioned above, B.t. or recombinant cellsexpressing a B.t. toxin can be treated to prolong the toxin activity andstabilize the cell. The pesticide microcapsule that is formed comprisesthe B.t. toxin within a cellular structure that has been stabilized andwill protect the toxin when the microcapsule is applied to theenvironment of the target pest. Suitable host cells may include eitherprokaryotes or eukaryotes, normally being limited to those cells whichdo not produce substances toxic to higher organisms, such as mammals.However, organisms which produce substances toxic to higher organismscould be used, where the toxic substances are unstable or the level ofapplication sufficiently low as to avoid any possibility of toxicity toa mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t.toxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability of protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as glutaraldehyde;anti-infectives, such as zephiran chloride and cetylpyridinium chloride;alcohols, such as isopropyl and ethanol; various histologic fixatives,such as Lugol iodine, Bouin's fixative, various acids and Helly'sfixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H.Freeman and Company, 1967); or a combination of physical (heat) andchemical agents that preserve and prolong the activity of the toxinproduced in the cell when the cell is administered to the hostenvironment. Examples of physical means are short wavelength radiationsuch as gamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. Methods for treatment of microbial cellsare disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

The cells generally will have enhanced structural stability which willenhance resistance to environmental conditions. Where the pesticide isin a proform, the method of cell treatment should be selected so as notto inhibit processing of the proform to the mature form of the pesticideby the target pest pathogen. For example, formaldehyde will crosslinkproteins and could inhibit processing of the proform of a polypeptidepesticide. The method of treatment should retain at least a substantialportion of the bio-availability or bioactivity of the toxin.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; survival in aqueous environments; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Growth of cells. The cellular host containing the B.t. insecticidal genemay be grown in any convenient nutrient medium, where the DNA constructprovides a selective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells of the invention can be cultured using standard art mediaand fermentation techniques. Upon completion of the fermentation cyclethe bacteria can be harvested by first separating the B.t. spores andcrystals from the fermentation broth by means well known in the art. Therecovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers, and othercomponents to facilitate handling and application for particular targetpests. These formulations and application procedures are all well knownin the art.

Formulations. Formulated bait granules containing an attractant andspores and crystals of the B.t. isolates, or recombinant microbescomprising the genes obtainable from the B.t. isolates disclosed herein,can be applied to the soil. Formulated product can also be applied as aseed-coating or root treatment or total plant treatment at later stagesof the crop cycle. Plant and soil treatments of B.t. cells maybeemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1-95% by weight of the pesticide while the liquid formulations willgenerally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g.,soil and foliage, by spraying, dusting, sprinkling, or the like.

Mutants. Mutants of the isolates of the invention can be made byprocedures well known in the art. For example, an asporogenous mutantcan be obtained through ethylmethane sulfonate (EMS) mutagenesis of anisolate. The mutants can be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

A smaller percentage of the asporogenous mutants will remain intact andnot lyse for extended fermentation periods; these strains are designatedlysis minus (−). Lysis minus strains can be identified by screeningasporogenous mutants in shake flask media and selecting those mutantsthat are still intact and contain toxin crystals at the end of thefermentation. Lysis minus strains are suitable for a cell treatmentprocess that will yield a protected, encapsulated toxin protein.

To prepare a phage resistant variant of said asporogenous mutant, analiquot of the phage lysate is spread onto nutrient agar and allowed todry. An aliquot of the phage sensitive bacterial strain is then plateddirectly over the dried lysate and allowed to dry. The plates areincubated at 30° C. The plates are incubated for 2 days and, at thattime, numerous colonies could be seen growing on the agar. Some of thesecolonies are picked and subcultured onto nutrient agar plates. Theseapparent resistant cultures are tested for resistance by cross streakingwith the phage lysate. A line of the phage lysate is streaked on theplate and allowed to dry. The presumptive resistant cultures are thenstreaked across the phage line. Resistant bacterial cultures show nolysis anywhere in the streak across the phage line after overnightincubation at 30° C. The resistance to phage is then reconfirmed byplating a lawn of the resistant culture onto a nutrient agar plate. Thesensitive strain is also plated in the same manner to serve as thepositive control. After drying, a drop of the phage lysate is placed inthe center of the plate and allowed to dry. Resistant cultures showed nolysis in the area where the phage lysate has been placed afterincubation at 30° C. for 24 hours.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Culturing of B.t. Isolates of the Invention

A subculture of the B.t. isolates, or mutants thereof, can be used toinoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone  7.5 g/l Glucose  1.0 g/l KH₂PO₄  3.4 g/l K₂HPO₄ 4.35 g/lSalt Solution 5.0 ml/l CaCl₂ Solution 5.0 ml/l pH 7.2 Salts Solution(100 ml) MgSO₄.7H₂O 2.46 g MnSO₄.H₂O 0.04 g ZnSO₄.7H₂O 0.28 g FeSO₄.7H₂O0.40 g CaCl₂ Solution (100 ml) CaCl₂.2H₂O 3.66 g

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and/or crystals, obtained in the above fermentation, canbe isolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Protein Purification for 45 kDa 80JJ1 Protein

One gram of lyophilized powder of 80JJ1 was suspended in 40 ml of buffercontaining 80 mM Tris-Cl pH 7.8, 5 mM EDTA, 100 μM PMSF, 0.5 μg/mlLeupeptin, 0.7. μg/ml Pepstatin, and 40 μg/ml Bestatin. The suspensionwas centrifuged, and the resulting supernatant was discarded. The pelletwas washed five times using 35-40 ml of the above buffer for each wash.The washed pellet was resuspended in 10 ml of 40% NaBr, 5 mM EDTA, 100μM PMSF, 0.5 μg/ml Leupeptin, 0.7 μg/ml Pepstatin, and 40 μg/ml Bestatinand placed on a rocker platform for 75 minutes. The NaBr suspension wascentrifuged, the supernatant was removed, and the pellet was treated asecond time with 40% NaBr, 5 mM EDTA, 100 μM PMSF, 0.5 μg/ml Leupeptin,0.7 μg/ml Pepstatin, and 40 μg/ml Bestatin as above. The supernatants(40% NaBr soluble) were combined and dialyzed against 10 mM CAPS pH10.0, 1 mM EDTA at 4° C. The dialyzed extracts were centrifuged and theresulting supernatant was removed. The pellet (40% NaBr dialysis pellet)was suspended in 5 ml of H₂O and centrifuged. The resultant supernatantwas removed and discarded. The washed pellet was washed a second time in10 ml of H₂O and centrifuged as above. The washed pellet was suspendedin 1.5 ml of H₂O and contained primarily three peptides with molecularweights of approximately 47 kDa, 45 kDa, and 15 kDa when analyzed usingSDS-PAGE. At this stage of purification, the suspended 40% NaBr dialysispellet contained approximately 21 mg/ml of protein by Lowry assay.

The peptides in the pellet suspension were separated using SDS-PAGE(Laemlli, U.K. [1970] Nature 227:680) in 15% acrylamide gels. Theseparated proteins were then electrophoretically blotted to a PVDFmembrane (Millipore Corp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 Vconstant. After one hour the PVDF membrane was rinsed in water brieflyand placed for 3 minutes in 0.25% Coomassie blue R-250, 50% methanol, 5%acetic acid. The stained membrane was destained in 40% MeOH, 5% aceticacid. The destained membrane was air-dried at room temperature (LeGendreet al. [1989] In A Practical Guide to Protein Purification ForMicrosequencing, P. Matsudaira, ed., Academic Press, New York, N.Y.).The membrane was sequenced using automated gas phase Edman degradation(Hunkapillar, M. W., R. M. Hewick, W. L. Dreyer, L. E. Hood [1983] Meth.Enzymol. 91:399).

The amino acid analysis revealed that the N-terminal sequence of the 45kDa band was as follows: Met-Leu-Asp-Thr-Asn (SEQ ID NO. 1).

The 47 kDa band was also analyzed and the N-terminal amino acid sequencewas determined to be the same 5-amino acid sequence as SEQ ID NO. 1.Therefore, the N-terminal amino acid sequences of the 47 kDa peptide andthe 45 kDa peptide were identical.

This amino acid sequence also corresponds to a sequence obtained from a45 kDa peptide obtained from PS80JJ1 spore/crystal powders, usinganother purification protocol, with the N-terminal sequence as follows:Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-Ile-Ser-Asn-Leu-Ala-Asn-Gly-Leu-Tyr-Thr-Ser-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO. 2).

EXAMPLE 3 Purification of the 14 kDa Peptide of PS80JJ1

0.8 ml of the white dialysis suspension (approximately 21 mg/ml)containing the 47 kDa, 45 kDa, and 15 kDa peptides, was dissolved in 10ml of 40% NaBr, and 0.5 ml of 100 mM EDTA were added. After about 18hours (overnight), a white opaque suspension was obtained. This wascollected by centrifugation and discarded. The supernatant wasconcentrated in a Centricon-30 (Amicon Corporation) to a final volume ofapproximately 15 ml. The filtered volume was washed with water by filterdialysis and incubated on ice, eventually forming a milky whitesuspension. The suspension was centrifuged and the pellet andsupernatant were separated and retained. The pellet was then suspendedin 1.0 ml water (approximately 6 mg/ml). The pellet containedsubstantially pure 15 kDa protein when analyzed by SDS-PAGE.

The N-terminal amino acid sequence was determined to be:Ser-Ala-Arg-Glu-Val-His-Ile-Glu-Ile-Asn-Asn-Thr-Arg-His-Thr-Leu-Gln-Leu-Glu-Ala-Lys-Thr-Lys-Leu(SEQ ID NO. 3).

EXAMPLE 4 Protein Purification and Characterization of PS149B1 45 kDaProtein

The P1 pellet was resuspended with two volumes of deionized water perunit wet weight, and to this was added nine volumes of 40% (w/w) aqueoussodium bromide. This and all subsequent operations were carried out onice or at 4-6° C. After 30 minutes, the suspension was diluted with 36volumes of chilled water and centrifuged at 25,000×g for 30 minutes togive a pellet and a supernatant.

The resulting pellet was resuspended in 1-2 volumes of water and layeredon a 20-40% (w/w) sodium bromide gradient and centrifuged at 8,000×g for100 minutes. The layer banding at approximately 32% (w/w) sodium bromide(the “inclusions”, or INC) was recovered and dialyzed overnight againstwater using a dialysis membrane with a 6-8 kDa MW cut-off. Particulatematerial was recovered by centrifugation at 25,000×g, resuspended inwater, and aliquoted and assayed for protein by the method of Lowry andby SDS-PAGE.

The resulting supernatant was concentrated 3- to 4-fold usingCentricon-10 concentrators, then dialyzed overnight against water usinga dialysis membrane with a 6-8 kDa MW cut-off. Particulate material wasrecovered by centrifugation at 25,000×g, resuspended in water, andaliquoted and assayed for protein by the method of Lowry and bySDS-PAGE. This fraction was denoted as P1.P2.

The peptides in the pellet suspension were separated using SDS-PAGE(Laemlli, U.K., supra) in 15% acrylamide gels. The separated proteinswere then electrophoretically blotted to a PVDF membrane (MilliporeCorp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 V constant. After one hourthe PVDF membrane was rinsed in water briefly and placed for 3 minutesin 0.25% Coomassie blue R-250, 50% methanol, 5% acetic acid. The stainedmembrane was destained in 40% MeOH, 5% acetic acid. The destainedmembrane was air-dried at room temperature (LeGendre et al., supra). Themembrane was sequenced using automated gas phase Edman degradation(Hunkapillar et al., supra).

Protein analysis indicated the presence of two major polypeptides, withmolecular weights of 47 kDa and 14 kDa. Molecular weights were measuredagainst standard polypeptides of known molecular weight. This processprovides only an estimate of true molecular weight. The 47 kDa band fromPS149B1 migrated on SDS-PAGE in a manner indistinguishable from the 47kDa protein from PS80JJ1. Likewise, the 14 kDa band from PS149B1migrated on SDS-PAGE in a manner indistinguishable from 14 kDa bandsfrom PS167H2 and PS80JJ1. Apart from these two polypeptides, which wereestimated to account for 25-35% (47 kDa) and 35-55% (15 kDa) of theCoomassie staining material respectively, there may be minor bands,including those of estimated MW at 46 kDa, 130 kDa, and 70 kDa.

Protein analysis indicated that fraction INC contained a singlepolypeptide with MW of 47 kDa, and that fraction P1.P2 contained asingle polypeptide with MW of 14 kDa. These polypeptides were recoveredin yields greater than 50% from P1.

The N-terminal amino acid sequence for the purified 47 kDa protein fromPS149B1 is:Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-Ile-Ser-Asn-His-Ala-Asn-Gly-Leu-Tyr-Ala-Ala-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO. 4).

The N-terminal amino acid sequence for the purified 14 kDa protein fromPS149B1 is:Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp-Lys-Thr-Lys-Leu-Asp-Gly-Gly-Arg-Trp-Arg-Thr-Ser-Pro-Xaa-Asn-Val-Ala-Asn-Asp-Gln-Ile-Lys-Thr-Phe-Val-Ala-Glu-Ser-Asn(SEQ ID NO. 5).

EXAMPLE 5 Amino Acid Sequence for 45 kDa and 14 kDa Toxins of PS167H2

The N-terminal amino acid sequence for the purified 45 kDa protein fromPS167H2 is:Met-Leu-Asp-Thr-Asn-Lys-Ile-Tyr-Glu-Ile-Ser-Asn-Tyr-Ala-Asn-Gly-Leu-His-Ala-Ala-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO. 6).

The N-terminal amino acid sequence for the purified 14 kDa protein fromPS167H2 is:Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp-Lys-Thr-Lys-Leu(SEQ ID NO. 7).

These amino acid sequences can be compared to the sequence obtained forthe 47 kDa peptide obtained from 80JJ1 spore/crystal powders with theN-terminal sequence (SEQ ID NO. 1) and to the sequence obtained for the14 kDa peptide obtained from 80JJ1 spore/crystal powders with theN-terminal sequence (SEQ ID NO. 3).

Clearly, the 45-47 kDa proteins are highly related and probablyrepresent one gene family, and the 14 kDa proteins are highly relatedand probably represent another gene family.

EXAMPLE 6 Molecular Cloning, Expression, and DNA Sequence Analysis of aNovel δ-Endotoxin Gene from Bacillus thuringiensis Strain PS80JJ1

Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cellsgrown to an optical density, at 600 nm, of 1.0. Cells were pelleted bycentrifugation and resuspended in protoplast buffer (20 mg/ml lysozymein 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After incubationat 37° C. for 1 hour, protoplasts were lysed by two cycles of freezingand thawing. Nine volumes of a solution of 0.1 M NaCl, 0.1% SDS, 0.1 MTris-Cl were added to complete lysis. The cleared lysate was extractedtwice with phenol:chloroform (1:1). Nucleic acids were precipitated withtwo volumes of ethanol and pelleted by centrifugation. The pellet wasresuspended in TE buffer and RNase was added to a final concentration of50 μg/ml. After incubation at 37° C. for 1 hour, the solution wasextracted once each with phenol:chloroform (1:1) and TE-saturatedchloroform. DNA was precipitated from the aqueous phase by the additionof one-tenth volume of 3 M NaOAc and two volumes of ethanol. DNA waspelleted by centrifugation, washed with 70% ethanol, dried, andresuspended in TE buffer.

An oligonucleotide probe for the gene encoding the PS80JJ1 45 kDa toxinwas designed from N-terminal peptide sequence data. The sequence of the29-base oligonucleotide probe was:

-   5′-ATG YTW GAT ACW AAT AAA GTW TAT GAA AT-3′ (SEQ ID NO. 8)    This oligonucleotide was mixed at four positions as shown. This    probe was radiolabeled with ³²P and used in standard condition    hybridization of Southern blots of PS80JJ1 total cellular DNA    digested with various restriction endonucleases. Representative    autoradiographic data from these experiments showing the sizes of    DNA restriction fragments containing sequence homology to the 44.3    kDa toxin oligonucleotide probe of SEQ ID NO. 8 are presented in    Table 3.

TABLE 3 RFLP of PS80JJ1 cellular DNA fragments on Southern blots thathybridized under standard conditions with the 44.3 kDa toxin geneoligonucleotide probe (SEQ ID NO. 8) Restriction Enzyme ApproximateFragment Size (kbp) EcoRI 6.0 HindIII 8.3 KpnI 7.4 PstI 11.5 XbaI 9.1These DNA fragments identified in these analyses contain all or asegment of the PS80JJ1 45 kDa toxin gene. The approximate sizes of thehybridizing DNA fragments in Table 3 are in reasonable agreement withthe sizes of a subset of the PS80JJ1 fragments hybridizing with aPS80JJ1 45 kDa toxin subgene probe used in separate experiments, aspredicted (see Table 4, below).

A gene library was constructed from PS80JJ1 DNA partially digested withSau3AI. Partial restriction digests were fractionated by agarose gelelectrophoresis. DNA fragments 9.3 to 23 kbp in size were excised fromthe gel, electroeluted from the gel slice, purified on an Elutip-D ionexchange column (Schleicher and Schuell, Keene, N.H.), and recovered byethanol precipitation. The Sau3AI inserts were ligated intoBamHI-digested LambdaGem-11 (Promega, Madison, Wis.). Recombinant phagewere packaged and plated on E. coli KW251 cells. Plaques were screenedby hybridization with the oligonucleotide probe described above.Hybridizing phage were plaque-purified and used to infect liquidcultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

Southern blot analysis revealed that one of the recombinant phageisolates contained an approximately 4.8 kbp XbaI-SacI band thathybridized to the PS80JJ1 toxin gene probe. The SacI site flanking thePS80JJ1 toxin gene is a phage vector cloning site, while the flankingXbaI site is located within the PS80JJ1 DNA insert. This DNA restrictionfragment was subcloned by standard methods into pBluescript S/K(Stratagene, San Diego, Calif.) for sequence analysis. The resultantplasmid was designated pMYC2421. The DNA insert was also subcloned intopHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised ofpBluescript S/K [Stratagene, La Jolla, Calif.] and the replicationorigin from a resident B.t. plasmid [D. Lereclus et al. (1989) FEMSMicrobiology Letters 60:211-218]) to yield pMYC2420.

An oligonucleotide probe for the gene encoding the PS80JJ1 14 kDa toxinwas designed from N-terminal peptide sequence data. The sequence of the28-base oligonucleotide probe was: 5′ GW GAA GTW CAT ATW GAA ATW AAT AATAC 3′ (SEQ ID NO. 29). This oligonucleotide was mixed at four positionsas shown. The probe was radiolabelled with ³²P and used in standardcondition hybridizations of Southern blots of PS80JJ1 total cellular andpMYC2421 DNA digested with various restriction endonucleases. These RFLPmapping experiments demonstrated that the gene encoding the 14 kDa toxinis located on the same genomic EcoRI fragment that contains theN-terminal coding sequence for the 44.3 kDa toxin.

To test expression of the PS80JJ1 toxin genes in B.t., pMYC2420 wastransformed into the acrystalliferous (Cry−) B.t. host, CryB (A.Aronson, Purdue University, West Lafayette, Ind.), by electroporation.Expression of both the approximately 14 and 44.3 kDa PS80JJ1 toxinsencoded by pMYC2420 was demonstrated by SDS-PAGE analysis. Toxin crystalpreparations from the recombinant CryB[pMYC2420] strain, MR536, wereassayed and found to be active against western corn rootworm.

The PS80JJ1 toxin genes encoded by pMYC2421 were sequenced using theABI373 automated sequencing system and associated software. The sequenceof the entire genetic locus containing both open reading frames andflanking nucleotide sequences is shown in SEQ ID NO. 30. The terminationcodon of the 14 kDa toxin gene is 121 base pairs upstream (5′) from theinitiation codon of the 44.3 kDa toxin gene (FIG. 2). The PS80JJ1 14 kDatoxin open reading frame nucleotide sequence (SEQ ID NO. 31), the 44.3kDa toxin open reading frame nucleotide sequence (SEQ ID NO. 10), andthe respective deduced amino acid sequences (SEQ ID NO. 32 and SEQ IDNO. 11) are novel compared to other toxin genes encoding pesticidalproteins.

Thus, the nucleotide sequence encoding the 14 kDa toxin of PS80JJ1 isshown in SEQ ID NO. 31. The deduced amino acid sequence of the 14 kDatoxin of PS80JJ1 is shown in SEQ ID NO. 32. The nucleotide sequencesencoding both the 14 and 45 kDa toxins of PS80JJ1, as well as theflanking sequences, are shown in SEQ ID NO. 30. The relationship ofthese sequences is shown in FIG. 2.

A subculture of E. coli NM522 containing plasmid pMYC2421 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 28, 1996. The accession number is NRRL B-21555.

EXAMPLE 7 RFLP and PCR Analysis of Additional Novel δ-Endotoxin Genesfrom Bacillus thuringiensis Strains PS149B1 and PS167H2

Two additional strains active against corn rootworm, PS149B1 andPS167H2, also produce parasporal protein crystals comprised in part ofpolypeptides approximately 14 and 45 kDa in size. Southern hybridizationand partial DNA sequence analysis were used to examine the relatednessof these toxins to the 80JJ1 toxins. DNA was extracted from these B.t.strains as described above, and standard Southern hybridizations wereperformed using the 14 kDa toxin oligonucleotide probe (SEQ ID NO. 29)and an approximately 800 bp PCR fragment of the 80JJ1 44.3 kDa toxingene-encoding sequence. Representative RFLP data from these experimentsshowing the sizes of DNA restriction fragments containing sequencehomology to the 44.3 kDa toxin are presented in Table 4. RepresentativeRFLP data from these experiments showing the sizes of DNA restrictionfragments containing sequence homology to the approximately 14 kDa toxinare presented in Table 5.

TABLE 4 RFLP of PS80JJ1, PS149B1, and PS167H2 cellular DNA fragments onSouthern blots that hybridized with the approximately 800 bp PS80JJ144.3 kDa toxin subgene probe under standard conditions Strain PS80JJ1PS149B1 PS167H2 Restriction enzyme Approximate fragment size (kbp) EcoRI6.4 5.7 2.6 1.3 2.8 0.6 HindIII 8.2 6.2 4.4 KpnI 7.8 10.0 11.5 PstI 12.09.2 9.2 8.2 XbaI 9.4 10.9 10.9 SacI 17.5 15.5 11.1 13.1 10.5 6.3

Each of the three strains exhibited unique RFLP patterns. Thehybridizing DNA fragments from PS149B1 or PS167H2 contain all or part oftoxin genes with sequence homology to the PS80JJ1 44.3 kDa toxin.

TABLE 5 Restriction fragment length polymorphisms of PS80JJ1, PS149B1,and PS167H2 cellular DNA fragments on Southern blots that hybridizedwith the PS80JJ1 14 kDa toxin oligonucleotide probe under standardconditions Strain PS80JJ1 PS149B1 PS167H2 Restriction enzyme Approximatefragment size (kbp) EcoRI 5.6 2.7 2.7 HindIII 7.1 6.0 4.7 XbaI 8.4 11.211.2

Each of the three strains exhibited unique RFLP patterns. Thehybridizing DNA fragments from PS149B1 or PS167H2 contain all or part oftoxin genes with sequence homology to the PS80JJ1 14 kDa toxin gene.

Portions of the toxin genes in PS149B1 or PS167H2 were amplified by PCRusing forward and reverse oligonucleotide primer pairs designed based onthe PS80JJ1 44.3 kDa toxin gene sequence. For PS149B1, the followingprimer pair was used:

Forward:

-   5′-ATG YTW GAT ACW AAT AAA GTW TAT GAA AT-3′ (SEQ ID NO. 8)

Reverse:

-   5′-GGA TTA TCT ATC TCT GAG TGT TCT TG-3′ (SEQ ID NO. 9)    For PS167H2, the same primer pair was used. These PCR-derived    fragments were sequenced using the ABI373 automated sequencing    system and associated software. The partial gene and peptide    sequences obtained are shown in SEQ ID NO. 12-15. These sequences    contain portions of the nucleotide coding sequences and peptide    sequences for novel corn rootworm-active toxins present in B.t.    strains PS149B1 or PS167H2.

EXAMPLE 8 Molecular Cloning and DNA Sequence Analysis of Novelδ-Endotoxin Genes from Bacillus thuringiensis Strains PS149B1 andPS167H2

Total cellular DNA was extracted from strains PS149B1 and PS167H2 asdescribed for PS80JJ1. Gene libraries of size-fractionated Sau3A partialrestriction fragments were constructed in Lambda-Gem11 for eachrespective strain as previously described. Recombinant phage werepackaged and plated on E. coli KW251 cells. Plaques were screened byhybridization with the oligonucleotide probe specific for the 44 kDatoxin gene. Hybridizing phage were plaque-purified and used to infectliquid cultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

For PS167H2, Southern blot analysis revealed that one of the recombinantphage isolates contained an approximately 4.0 to 4.4 kbp HindIII bandthat hybridized to the PS80JJ1 44 kDa toxin gene 5′ oligonucleotideprobe (SEQ ID NO. 8). This DNA restriction fragment was subcloned bystandard methods into pBluescript S/K (Stratgene, San Diego, Calif.) forsequence analysis. The fragment was also subcloned into the high copynumber shuttle vector, pHT370 (Arantes, O., D. Lereclus [1991] Gene108:115-119) for expression analyses in Bacillus thuringiensis (seebelow). The resultant recombinant, high copy number bifunctional plasmidwas designated pMYC2427.

The PS167H2 toxin genes encoded by pMYC2427 were sequenced using the ABIautomated sequencing system and associated software. The sequence of theentire genetic locus containing both open reading frames and flankingnucleotide sequences is shown in SEQ ID NO. 34. The termination codon ofthe 14 kDa toxin gene is 107 base pairs upstream (5′) from theinitiation codon of the 44 kDa toxin gene. The PS167H2 14 kDa toxincoding sequence (SEQ ID NO. 35), the 44 kDa toxin coding sequence (SEQID NO. 37), and the respective deduced amino acid sequences, SEQ ID NO.36 and SEQ ID NO. 38, are novel compared to other known toxin genesencoding pesticidal proteins. The toxin genes are arranged in a similarmanner to, and have some homology with, the PS80JJ1 14 and 44 kDatoxins.

A subculture of E. coli NM522 containing plasmid pMYC2427 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 26, 1997. The accession number is NRRL B-21672.

For PS149B1, Southern blot analysis using the PS80JJ1 44 kDaoligonucleotide 5′ probe (SEQ ID NO. 8) demonstrated hybridization of anapproximately 5.9 kbp ClaI DNA fragment. Complete ClaI digests ofPS149B1 genomic DNA were size fractionated on agarose gels and clonedinto pHTBlueII. The fragment was also subcloned into the high copynumber shuttle vector, pHT370 (Arantes, O., D. Lereclus [1991] Gene108:115-119) for expression analyses in Bacillus thuringiensis (seebelow). The resultant recombinant, high copy number bifunctional plasmidwas designated pMYC2429.

The PS149B1 toxin genes encoded by pMYC2429 were sequenced using the ABIautomated sequencing system and associated software. The sequence of theentire genetic locus containing both open reading frames and flankingnucleotide sequences is shown in SEQ ID NO. 39. The termination codon ofthe 14 kDa toxin gene is 108 base pairs upstream (5′) from theinitiation codon of the 44 kDa toxin gene. The PS149B1 14 kDa toxincoding sequence (SEQ ID NO. 40), the 44 kDa toxin coding sequence (SEQID NO. 42), and the respective deduced amino acid sequences, SEQ ID NO.41 and SEQ ID NO. 43, are novel compared to other known toxin genesencoding pesticidal proteins. The toxin genes are arranged in a similarmanner as, and have some homology with, the PS80JJ1 and PS167H2 14 and44 kDa toxins. Together, these three toxin operons comprise a new familyof pesticidal toxins.

A subculture of E. coli NM522 containing plasmid pMYC2429 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 26, 1997. The accession number is NRRL B-21673.

EXAMPLE 9 PCR Amplification for Identification and Cloning Novel CornRootworm-Active Toxin

The DNA and peptide sequences of the three novel approximately 45 kDacorn rootworm-active toxins from PS80JJ1, PS149B1, and PS167H2 (SEQ IDNOS. 12-15) were aligned with the Genetics Computer Group sequenceanalysis program Pileup using a gap weight of 3.00 and a gap lengthweight of 0.10. The sequence alignments were used to identify conservedpeptide sequences to which oligonucleotide primers were designed thatwere likely to hybridize to genes encoding members of this novel toxinfamily. Such primers can be used in PCR to amplify diagnostic DNAfragments for these and related toxin genes. Numerous primer designs tovarious sequences are possible, four of which are described here toprovide an example. These peptide sequences are:

Asp-Ile-Asp-Asp-Tyr-Asn-Leu (SEQ ID NO. 16) Trp-Phe-Leu-Phe-Pro-Ile-Asp(SEQ ID NO. 17) Gln-Ile-Lys-Thr-Thr-Pro-Tyr-Tyr (SEQ ID NO. 18)Tyr-Glu-Trp-Gly-Thr-Glu (SEQ ID NO. 19)The corresponding nucleotide sequences are:

5′-GATATWGATGAYTAYAAYTTR-3′ (SEQ ID NO. 20) 5′-TGGTTTTTRTTTCCWATWGAY-3′(SEQ ID NO. 21) 5′-CAAATHAAAACWACWCCATATTAT-3′ (SEQ ID NO. 22)5′-TAYGARTGGGGHACAGAA-3′. (SEQ ID NO. 23)Forward primers for polymerase amplification in thermocycle reactionswere designed based on the nucleotide sequences of SEQ ID NOS. 20 and21.

Reverse primers were designed based on the reverse complement of SEQ IDNOS. 22 and 23:

5′-ATAATATGGWGTWGTTTTDATTTG-3′ (SEQ ID NO. 24) 5′-TTCTGTDCCCCAYTCRTA-3′.(SEQ ID NO. 25)These primers can be used in combination to amplify DNA fragments of thefollowing sizes (Table 6) that identify genes encoding novel cornrootworm toxins.

TABLE 6 Predicted sizes of diagnostic DNA fragments (base pairs)amplifiable with primers specific for novel corn rootworm-active toxinsPrimer pair (SEQ ID NO.) DNA fragment size (bp) 20 + 24 495 20 + 25 59421 + 24 471 21 + 25 580

Similarly, entire genes encoding novel corn rootworm-active toxins canbe isolated by polymerase amplification in thermocycle reactions usingprimers designed based on DNA sequences flanking the open readingframes. For the PS80JJ1 44.3 kDa toxin, one such primer pair wasdesigned, synthesized, and used to amplify a diagnostic 1613 bp DNAfragment that included the entire toxin coding sequence. These primersare:

Forward: 5′-CTCAAAGCGGATCAGGAG-3′ (SEQ ID NO. 26) Reverse:5′-GCGTATTCGGATATGCTTGG-3′. (SEQ ID NO. 27)For PCR amplification of the PS80JJ1 14 kDa toxin, the oligonucleotidecoding for the N-terminal peptide sequence (SEQ ID NO. 29) can be usedin combination with various reverse oligonucleotide primers based on thesequences in the PS80JJ1 toxin gene locus. One such reverse primer hasthe following sequence:

-   5′ CATGAGATTTATCTCCTGATCCGC 3′ (SEQ ID NO. 33).    When used in standard PCR reactions, this primer pair amplified a    diagnostic 1390 bp DNA fragment that includes the entire 14 kDa    toxin coding sequence and some 3′ flanking sequences corresponding    to the 121 base intergenic spacer and a portion of the 44.3 kDa    toxin gene. When used in combination with the 14 kDa forward primer,    PCR will generate a diagnostic 322 base pair DNA fragment.

EXAMPLE 10 Bioassay of Protein

A preparation of the insoluble fraction from the dialyzed NaBr extractof 80JJ1 containing the 47 kDa, 45 kDa, and 15 kDa peptides wasbioassayed against Western corn rootworm and found to exhibitsignificant toxin activity.

EXAMPLE 11 Bioassay of Protein

The purified protein fractions from PS149B1 were bioassayed againstwestern corn rootworm and found to exhibit significant toxin activitywhen combined. In fact, the combination restored activity to that notedin the original preparation (P1). The following bioassay data setpresents percent mortality and demonstrates this effect.

TABLE 7 Concentration (μg/cm²) P1 INC P1.P2 INC + P1.P2 300 88, 100, 9419 13 100 100 94, 50, 63 31 38 94 33.3 19, 19, 44 38 13 50 11.1 13, 56,25 12 31 13 3.7 0, 50, 0 0 31 13 1.2 13, 43, 12 0 12 19 0.4 6, 12, 6 2519 6

EXAMPLE 12 Clone Dose-Response Bioassays

The PS80JJ1 toxin operon was also subcloned from pMYC2421 into pHT370for direct comparison of bioactivity with the recombinant toxins clonedfrom PS 149B1 and PS167H2. The resultant recombinant, high copy numberbifunctional plasmid was designated pMYC2426.

A subculture of E. coli NM522 containing plasmid pMYC2426 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 26, 1997. The accession number is NRRL B-21671.

To test expression of the PS80JJ1, PS149B1 and PS167H2 toxin genes inB.t., pMYC2426, pMYC2427 and pMYC2429 were separately transformed intothe acrystalliferous (Cry−) B.t. host, CryB (A. Aronson, PurdueUniversity, West Lafayette, Ind.), by electroporation. The recombinantstrains were designated MR543 (CryB [pMYC2426]), MR544 (CryB [pMYC2427])and MR546 (CryB [pMYC2429]), respectively. Expression of both theapproximately 14 and 44 kDa toxins was demonstrated by SDS-PAGE analysisfor each recombinant strain.

Toxin crystal preparations from the recombinant strains were assayedagainst western corn rootworm. Their diet was amended with sorbic acidand SIGMA pen-strep-ampho-B. The material was top-loaded at a rate of 50μl of suspension per cm² diet surface area. Bioassays were run withneonate Western corn rootworm larvae for 4 days at approximately 25° C.Percentage mortality and top-load LC₅₀ estimates for the clones(pellets) are set forth in Table 8.

TABLE 8 Percentage mortality at given protein concentration (μg/cm²)Sample 50 5 0.5 MR543 pellet 44 19 9 MR544 pellet 72 32 21 MR546 pellet52 32 21 dH2O 7

EXAMPLE 13 Insertion and Expression of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants withgenes encoding the insecticidal toxin. The transformed plants areresistant to attack by the target pest.

The novel corn rootworm-active genes described here can be optimized forexpression in other organisms. Maize optimized gene sequences encodingthe 14 and 44 kDa PS80JJ1 toxins are disclosed in SEQ ID NO. 44 and SEQID NO. 45, respectively.

Genes encoding pesticidal toxins, as disclosed herein, can be insertedinto plant cells using a variety of techniques which are well known inthe art. For example, a large number of cloning vectors comprising areplication system in E. coli and a marker that permits selection of thetransformed cells are available for preparation for the insertion offoreign genes into higher plants. The vectors comprise, for example,pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, thesequence encoding the B.t. toxin can be inserted into the vector at asuitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120516; Hoekema(1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci.4:1-46; and An et al. (1985) EMBO J. 4:277-287.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831, which is herebyincorporated by reference. Also, advantageously, plants encoding atruncated toxin will be used. The truncated toxin typically will encodeabout 55% to about 80% of the full length toxin. Methods for creatingsynthetic B.t. genes for use in plants are known in the art.

EXAMPLE 14 Cloning of B.t. Genes Into Insect Viruses

A number of viruses are known to infect insects. These viruses include,for example, baculoviruses and entomopoxyiruses. In one embodiment ofthe subject invention, genes encoding the insecticidal toxins, asdescribed herein, can be placed within the genome of the insect virus,thus enhancing the pathogenicity of the virus. Methods for constructinginsect viruses which comprise B.t. toxin genes are well known andreadily practiced by those skilled in the art. These procedures aredescribed, for example, in Merryweather et al. (Merryweather, A. T., U.Weyer, M. P. G. Harris, M. Hirst, T. Booth, R. D. Possee (1990) J. Gen.Virol. 71:1535-1544) and Martens et al (Martens, J. W. M., G. Honee, D.Zuidema, J. W. M. van Lent, B. Visser, J. M. Vlak (1990) Appl.Environmental Microbiol. 56(9):2764-2770).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

1. An isolated polynucleotide that encodes a protein that has toxinactivity against a corn rootworm pest, wherein the full complement ofthe nucleotide sequence represented by SEQ ID NO:31 hybridizes understringent conditions with a nucleic acid sequence that codes for saidprotein.
 2. The polynucleotide of claim 1 wherein said protein has amolecular weight of between about 10 kDa and about 15 kDa. 3.Thepolynucleotide of claim 1 wherein said stringent conditions comprise awash in 1×SSPE and 0.1% SDS at room temperature.
 4. The polynucleotideof claim 1 wherein said stringent conditions comprise a wash in 0.2×SSPEand 0.1% SDS at room temperature.
 5. An isolated polynucleotide thatencodes a protein that has toxin activity against a corn rootworm pest,wherein the frill complement of a nucleotide sequence selected from thegroup consisting of SEQ ID NO:35, SEQ ID NO:40, and SEQ ID NO:44hybridizes under stringent conditions with a nucleic acid sequence thatcodes for said protein.
 6. The polynucleotide of claim 5 wherein saidnucleotide sequence is SEQ ID NO:35.
 7. The polynucleotide of claim 5wherein said nucleotide sequence is SEQ ID NO:40.
 8. The polynucleotideof claim 5 wherein said nucleotide sequence is SEQ ID NO:44.
 9. Anisolated polynucleotide that encodes a protein that has toxin activityagainst a corn rootworm pest, wherein the full complement of anucleotide sequence that codes for the amino acid sequence representedby SEQ ID NO:32 hybridizes under stringent conditions with a nucleicacid sequence that codes for said protein.
 10. The polynucleotide ofclaim 9 wherein said protein has a molecular weight of between about 10kDa and about 15 kDa.
 11. The polynucleotide of claim 9 wherein saidstringent conditions comprise a wash in 1×SSPE and 0.1% SDS at roomtemperature.
 12. The polynucleotide of claim 9 wherein said stringentconditions comprise a wash in 0.2×SSPE and 0.1% SDS at room temperature.13. An isolated polynucleotide that encodes a protein that has toxinactivity against a corn rootworm pest, wherein the full complement of anucleotide sequence that codes for the amino acid sequence selected fromthe group consisting of SEQ ID NO:36 and SEQ ID NQ:41 hybridizes understringent conditions with a nucleic acid sequence that codes for saidprotein.
 14. The polynucleotide of claim 13 wherein said amino acidsequence is SEQ ID NO:36.
 15. The polynucleotide of claim 13 whereinsaid amino acid sequence is SEQ ID NO:41.
 16. A transgenic cellcomprising an isolated polynucleotide of claim
 1. 17. A transgenic cellcomprising an isolated polynucleotide of claim
 5. 18. A transgenic cellcomprising an isolated polynucleotide of claim
 9. 19. A transgenic cellcomprising an isolated polynucleotide of claim
 13. 20. The transgeniccell of claim 16 wherein said cell is a microbial cell.
 21. Thetransgenic cell of claim 16 wherein said cell is a bacterial cell. 22.The transgenic cell of claim 16 wherein said cell is a plant cell. 23.The transgenic cell of claim 16 wherein said cell is a cell of a cornplant.
 24. The transgenic cell of claim 16 wherein said cell is a cornroot cell.